This application is a national stage filing of PCT/IB98/00270, filed on Feb. 23, 1998, under 35 U.S.C. xc2xa7371.
The present invention relates to a method of inhibiting gene expression, particularly inhibiting gene expression in a plant. The present invention also relates to a nucleotide sequence useful in the method. In addition, the present invention relates to a promoter that is useful for expressing the nucleotide sequence.
Starch is one of the main storage carbohydrates in plants, especially higher plants. The structure of starch consists of amylose and amylopectin. Amylose consists essentially of straight chains of xcex1-1-4-linked glycosyl residues. Amylopectin comprises chains of xcex1-1-4-linked glycosyl residues with some xcex1-1-6 branches. The branched nature of amylopectin is accomplished by the action of inter alia an enzyme commonly known as the starch branching enzyme (xe2x80x9cSBExe2x80x9d). SBE catalyses the formation of branch points in the amylopectin molecule by adding xcex1-1,4 glucans through xcex1-1,6-glucosidic branching linkages. The biosynthesis of amylose and amylopectin is schematically shown in FIG. 1, whereas the xcex1-1-4-links and the xcex1-1-6 links are shown in FIG. 2.
In Potato, it is known that two classes of SBE exist. In our copending international patent applications PCT/EP96/03052 and PCT/EP96/03053, class B potato SBE and a gene encoding it are discussed. In international patent application WO96/34968, class A potato SBE and a cDNA encoding it are disclosed.
It is known that starch is an important raw material. Starch is widely used in the food, paper. and chemical industries. However, a large fraction of the starches used in these industrial applications are post-harvest modified by chemical, physical or enzymatic methods in order to obtain starches with certain required functional properties.
Within the past few years it has become desirable to make genetically modified plants which could be capable of producing modified starches which could be the same as the post-harvest modified starches. It is also known that it may be possible to prepare such genetically modified plants by expression of antisense nucleotide coding sequences. In this regard, June Bourque provides a detailed sunmmary of antisense strategies for the genetic nanipulations in plants (Bourque 1995 Plant Science 105 pp 125-149). At this stage, reference could be made to FIG. 3 which is a schematic diagram of one of the proposed mechanisms of antisense-RNA inhibition.
In particular, WO 92/11375 reports on a method of genetically modifying potato so as to form amylose-type starch. The method involves the use of an anti-sense construct that can apparently inhibit, to a varying extent, the expression of the gene coding for formation of the branching enzyme in potato. The antisense construct of WO 92/11375 consists of a tuber specific promoter, a transcription start sequence and the first exon of the branching. enzyme in antisense direction. However, WO 92/11375 does not provide any antisense sequence data. In addition, WO 92/11375 only discloses the use of the potato GBSS promoter.
WO 92/14827 reports on a plasmid that, after insertion into the genome of a plant, can apparently cause changes in the carbohydrate concentration and carbohydrate composition. such as the concentration and composition of amylose and amylopectin, in the regenerated plant. The plasmid contains part of the coding sequence of a branching enzyme in an antisense orientation.
EP-A-0647715 reports on the use of antisense endogenous mRNA coding DNA to alter the characteristics and the metabolic pathways of ornamental plants.
EP-A-0467349 reports on the expression of sequences that are antisense to sequences upstream of a promoter to control gene expression.
EP-A-0458367 and U.S. Pat. No. 5,107,065 report on the expression of a nucleotide sequence to regulate gene expression in a plant. The nucleotide sequence is complementary to a mRNA sequence of a gene and may cover all or a portion of the noncoding region of the gene. In other words, the nucleotide sequences of EP-A-0458367 and U.S. Pat. No. 5,107,065 must at least comprise a sequence that is complementary to a coding region. EP-A-0458367 and U.S. Pat. No. 5,107,065 contain minimal sequence information.
WO96/34968 discusses the use of antisense sequences complementary to sequences which encode class A and class B potato SBE to downregulate SBE expression in potato plants. The sequences used are complementary to SBE coding sequences.
Kuipers et al in Mol. Gen. Genet. [1995]246 745-755 report on the expression of a series of nucleotides that are antisense to part of the genomic intron sequences of potato granule bound starch synthetase. Here the antisense intron sequences are attached to a part of the antisense exon sequencesxe2x80x94wherein the intron sequences and the exon sequences are naturally associated with each other. In addition, the expressed antisense intron sequences are at most 231 bp in length.
Likewise, Kull et al in J. Genet and Breed. [1995] 49 69-76 report on the expression of a series of nucleotides that are antisense to part of the genomic intron sequences of potato granule bound starch synthetase. Likewise, here the antisense intron sequences are attached to a part of the antisense exon sequencesxe2x80x94wherein the intron sequences and the exon sequences are naturally associated with each other. In addition, likewise, the expressed antisense intron sequences are at most 231 bp in length.
Shimada et al in Theor. Appl. Genet. [1993]86 665-672 report on the expression of a series of nucleotides that are antisense to part of the genomic intron sequences of rice granule bound starch synthetase. Here the antisense intron sequences are attached to a part of the antisense exon sequencesxe2x80x94wherein the intron sequences and the exon sequences are naturally associated with each other. In addition, the expressed antisense intron sequences are less than 350 bp in length.
Reviews on how enzymatic activity can be affected by expression of particular nucleotide sequences may be found in the teachings of Finnegan and McElroy [1994] Biotechnology 12 883-888; and Matzke and Matzke [1995] TIG 11 1-3.
Whilst it is known that enzymatic activity can be affected by expression of particular nucleotide sequences there is still a need for a method that can more reliably and/or more efficiently and/or more specifically affect enzymatic activity.
According to a first aspect of the present invention there is provided a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence partially or completely codes for (is) an intron of the potato class A SBE gene in an antisense orientation optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in an antisense or sense orientation; and wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron.
According to a second aspect of the present invention there is provided a method of affecting enzymatic activity in a starch producing organism (or a cell, a tissue or an organ thereof) comprising expressing in the starch producing organism (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron of the potato class A SBE gene, in an antisense orientation optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in an antisense or sense orientation; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
Preferably, the class A SBE gene antisense intron construct is used in combination with a potato class B SBE gene antisense intron construct as defined in PCT/EP96/03052. However, it may also be used independently thereof, to target class A SBE alone, or in combination with other transgenes, to further manipulate starch quality in potato plants.
According to a third aspect of the present invention, therefore. there is provided an antisense sequence comprising the nucleotide sequence shown as any one of SEQ.I.D. No. 15 to SEQ.I.D. No. 27 and the complement of SEQ. ID. No.38, or a variant, derivative or homologue thereof.
According to a fourth aspect of the present invention there is provided a promoter comprising the sequence shown as SEQ.I.D. No. 14 or a variant, derivative or homologue thereof.
According to a fifth aspect of the present invention there is provided a construct capable of comprising or expressing the present invention.
According to a sixth aspect of the present invention there is provided a vector comprising or expressing the present invention.
According to a seventh aspect of the present invention there is provided a cell, tissue or organ comprising or expressing the present invention.
According to an eighth aspect of the present invention there is provided a transgenic starch producing organism comprising or expressing the present invention.
According to a ninth aspect of the present invention there is provided a starch obtained from the present invention.
According to a tenth aspect of the present invention there is provided pSS17 and pSS18.
According to an eleventh aspect of the present invention there is provided a nucleotide sequence that is antisense to any one or more of the intron sequences obtainable from class A SBE, and especially those obtainable from intron of class A SBE as set forth in SEQ. ID. No. 38.
A key advantage of the present invention is that it provides a method for preparing modified starches that is not dependent on the need for post-harvest modification of starches. Thus the method of the present invention obviates the need for the use of hazardous chemicals that are normally used in the post-harvest modification of starches.
In addition, the present invention provides inter alia genetically modified plants which are capable of producing modified and/or novel and/or improved starches whose properties would satisfy various industrial requirements.
Thus, the present invention provides a method of preparing tailor-made starches in plants which could replace the post-harvest modified starches.
Also, the present invention provides a method that enables modified starches to be prepared by a method that can have a more beneficial effect on the environment than the known post-harvest modification methods which are dependent on the use of hazardous chemicals and large quantities of energy.
An other key advantage of the present invention is that it provides a method that may more reliably and/or more efficiently and/or more specifically affect enzymatic activity when compared to the known methods of affecting enzymatic activity. With regard to this advantage of the present invention it is to be noted that there is some degree of homology between coding regions of SBEs. However, there is little or no homology with the intron sequences of SBEs.
Thus, antisense intron expression provides a mechanism to affect selectively the expression of a particular class A SBE. This advantageous aspect could be used, for example, to reduce or eliminate a particular SBE enzyme, especially a class A SBE enzyme, and replace that enzyme with another enzyme which can be another branching enzyme or even a recombinant version of the affected enzyme or even a hybrid enzyme which could for example comprise part of a SBE enzyme from one source and at least a part of another SBE enzyme from another source. This particular feature of the present invention is covered by the combination aspect of the present invention which is discussed in more detail later.
Thus the present invention provides a mechanism for selectively affecting class A SBE activity. This is in contrast to the prior art methods which are dependent on the use of for example antisense exon expression whereby it would not be possible to introduce new SBE activity without affecting that activity as well.
In the context of the present invention, class B SBE is synonymous with SBE I: class A SBE is synonymous with SBE II. Class A SBE is as defined in WO96/34968, incorporated herein by reference. Preferably, the antisense intron construct used comprises intron 1 of class A SBE, which is 2.0 kb in length and is located starting at residue 45 of the coding sequence of class A SEE. The boundaries of the intron may be calculated by searching for consensus intron boundary sequences. and are shown in attached FIG. 13. Class B SBE is substantially as defined in the sequences given herein and in PCT/EP96/03052.
Preferably with the first aspect of the present invention starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
Preferably with the second aspect of the present invention the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron.
Preferably with the fourth aspect of the present invention the promoter is in combination with a gene of interest (xe2x80x9cGOIxe2x80x9d).
Preferably the enzymatic activity is reduced or eliminated.
Preferably the nucleotide sequence codes for at least substantially all of at least one intron in an antisense orientation.
Preferably the nucleotide sequence codes, partially or completely, for two or more introns and wherein each intron is in an anti-sense orientation.
Preferably the nucleotide sequence comprises at least 350 nucleotides (e.g. at least 350 bp), more preferably at least 500 nucleotides (e.g. at least 500 bp).
Preferably the nucleotide sequence comprises the complement of the sequence shown in SEQ. ID. No. 38, or a fragment thereof.
Preferably the nucleotide sequence is expressed by a promoter having a sequence shown as SEQ. I.D. No 14 or a variant, derivative or homologue thereof.
Preferably the transgenic starch producing organism is a plant.
A preferred aspect of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
A more preferred aspect of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed; and wherein the nucleotide sequence comprises the sequence shown as any one of SEQ.I.D. No. 15 to SEQ.I.D. No. 27 or a variant, derivative or homologue thereof, including combinations thereof.
The term xe2x80x9cnucleotidexe2x80x9d in relation to the present invention includes DNA and RNA. Preferably it means DNA, more preferably DNA prepared by use of recombinant DNA techniques.
The term xe2x80x9cintronxe2x80x9d is used in its normal sense as meaning a segment of nucleotides, usually DNA, that is transcribed but does not encode part or all of an expressed protein or enzyme.
The term xe2x80x9cexonxe2x80x9d is used in its normal sense as meaning a segment of nucleotides, usually DNA, encoding part or all of an expressed protein or enzyme.
Thus, the term xe2x80x9cintronxe2x80x9d refers to gene regions that are transcribed into RNA molecules, but which are spliced out of the RNA before the RNA is translated into a protein. In contrast, the term xe2x80x9cexonxe2x80x9d refers to gene regions that are transcribed into RNA and subsequently translated into proteins.
The terms xe2x80x9cvariantxe2x80x9d or xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the respective nucleotide sequence providing the resultant nucleotide sequence can affect enzyme activity in a plant, or cell or tissue thereof, preferably wherein the resultant nucleotide sequence has at least the same effect as the complement of the sequence shown as SEQ.I.D. No. 38. In particular, the term xe2x80x9chomologuexe2x80x9d covers homology with respect to similarity of structure and/or similarity of function providing the resultant nucleotide sequence has the ability to affect enzymatic activity in accordance with the present invention. With respect to sequence homology (i.e. similarity), preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences.
Likewise, the terms xe2x80x9cvariantxe2x80x9d or xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the promoter of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the respective promoter sequence providing the resultant promoter sequence allows expression of a GOI, preferably wherein the resultant promoter sequence has at least the same effect as SEQ.I.D. No. 14. In particular, the term xe2x80x9chomologuexe2x80x9d covers homology with respect to similarity of structure and/or similarity of function providing the resultant promoter sequence has the ability to allow for expression of a GOI, such as a nucleotide sequence according to the present invention. With respect to sequence homology (i.e. similarity), preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences.
The term xe2x80x9cantisensexe2x80x9d means a nucleotide sequence that is complementary to, and can therefore hybridise with, any one or all of the intron sequences of the present invention, including partial sequences thereof.
With the present invention, the antisense intron can be complementary to an entire intron of the gene to be inhibited. However, in some circumstances, partial antisense sequences may be used (i.e. sequences that are not or do not comprise the full complementary sequence) providing the partial sequences affect enzymatic activity. Suitable examples of partial sequences include sequences that are shorter than the full complement of SEQ. ID. No. 38 but which comprise nucleotides that are at least antisense to the sense intron sequences adjacent the respective exon or exons.
With regard to the second aspect of the present invention (i.e. specifically affecting SBE activity), the nucleotide sequences of the present invention may comprise one or more sense or antisense exon sequences of the SBE gene, including complete or partial sequences thereof, providing the nucleotide sequences can affect SBE activity, preferably wherein the nucleotide sequences reduce or eliminate SBE activity. Preferably, the nucleotide sequence of the second aspect of the present invention does not comprise an antisense exon sequence.
The term xe2x80x9cvectorxe2x80x9d includes an expression vector and a transformation vector. The term xe2x80x9cexpression vectorxe2x80x9d means a construct capable of in vivo or in vitro expression. The term xe2x80x9ctransformation vectorxe2x80x9d means a construct capable of being transferred from one species to anotherxe2x80x94such as from an E. Coli plasmid to a fungus or a plant cell, or from an Agrobacterium to a plant cell.
The term xe2x80x9cconstructxe2x80x9dxe2x80x94which is synonymous with terms such as xe2x80x9cconjugatexe2x80x9d, xe2x80x9ccassettexe2x80x9d and xe2x80x9chybridxe2x80x9dxe2x80x94in relation to the antisense nucleotide sequence aspect of the present invention includes the nucleotide sequence according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term xe2x80x9cfusedxe2x80x9d in relation to the present invention which includes direct or indirect attachment. The terms do not cover the natural combination of the wild type SBE gene when associated with the wild type SBE gene promoter in their natural environment.
The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred. Various markers exist which may be used in, for example, plantsxe2x80x94such as mannose. Other examples of markers include those that provide for antibiotic resistancexe2x80x94e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
The construct of the present invention preferably comprises a promoter. The term xe2x80x9cpromoterxe2x80x9d is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression. Examples of suitable promoters are those that can direct efficient expression of the nucleotide sequence of the present invention and/or in a specific type of cell. Some examples of tissue specific promoters are disclosed in WO 92/11375.
The promoter could additionally include conserved regions such as a Pribnow Box or a TATA box. The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. Suitable examples of such sequences include the Shl-intron or an ADH intron. Other sequences include inducible elementsxe2x80x94such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5xe2x80x2 leader sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
As mentioned, the construct and/or the vector of the present invention may include a transcriptional initiation region which may provide for regulated or constitutive expression. Any suitable promoter may be used for the transcriptional initiation region, such as a tissue specific promoter. In one aspect, preferably the promoter is the patatin promoter or the E35S promoter. In another aspect, preferably the promoter is the SBE promoter.
If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, tuber, stem, sprout, root and leaf tissues, preferably tuber. By way of example, the promoter for the nucleotide sequence of the present invention can be the xcex1-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the xcex1-Amy 637 promoter) as described in our co-pending UK patent application No. 9421292.5 filed Oct. 21, 1994. Alternatively, the promoter for the nucleotide sequence of the present invention can be the xcex1-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the xcex1-Amy 351 promoter) as described in our co-pending UK patent application No. 9421286.7 filed Oct. 21, 1994.
The present invention also encompasses the use of a promoter to express a nucleotide sequence according to the present invention, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses the nucleotide sequence of the present invention in a more specific manner such as in just one specific tissue type or organ. The term xe2x80x9cinactivatedxe2x80x9d means partial inactivation in the sense that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter. However, as mentioned above, the modified promoter is capable of expressing a gene coding for the enzyme of the present invention in at least one (but not all) specific tissue of the original promoter. Examples of partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase. Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part. Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter.
The construct and/or the vector of the present invention may include a transcriptional termination region.
The nucleotide according to the present invention can be expressed in combination (but not necessarily at the same time) with an additional construct. Thus the present invention also provides a combination of constructs comprising a first construct comprising the nucleotide sequence according to the present invention operatively linked to a first promoter; and a second construct comprising a GOI operatively linked to a second promoter (which need not be the same as the first promoter). With this aspect of the present invention the combination of constructs may be present in the same vector, plasmid, cells, tissue, organ or organism. This aspect of the present invention also covers methods of expressing the same, preferably in specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a plant. With this aspect of the present invention the second construct does not cover the natural combination of the gene coding for an enzyme ordinarily associated with the wild type gene promoter when they are both in their natural environment.
An example of a suitable combination would be a first construct comprising the nucleotide sequence of the present invention and a promoter, such as the promoter of the present invention, and a second construct comprising a promoter, such as the promoter of the present invention, and a GOI wherein the GOI codes for another starch branching enzyme either in sense or antisense orientation.
The above comments relating to the term xe2x80x9cconstructxe2x80x9d for the antisense nucleotide aspect of the present invention are equally applicable to the term xe2x80x9cconstructxe2x80x9d for the promoter aspect of the present invention. In this regard, the term includes the promoter according to the present invention directly or indirectly attached to a GOI.
The term xe2x80x9cGOIxe2x80x9d with reference to the promoter aspect of the present invention or the combination aspect of the present invention means any gene of interest, which need not necessarily code for a protein or an enzymexe2x80x94as is explained later. A GOI can be any nucleotide sequence that is either foreign or natural to the organism in question, for example a plant.
Typical examples of a GOI include genes encoding for other proteins or enzymes that modify metabolic and catabolic processes. The GOI may code for an agent for introducing or increasing pathogen resistance.
The GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. An example of such a GOI is the nucleotide sequence according to the present invention.
The GOI may even code for a protein that is non-natural to the host organismxe2x80x94e.g. a plant. The GOI may code for a compound that is of benefit to animals or humans. For example, the GOI could code for a pharmaceutically active protein or enzyme such as any one of the therapeutic compounds insulin, interferon, human serum albumin, human growth factor and blood clotting factors. The GOI may even code for a protein giving additional nutritional value to a food or feed or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant). The GOI may even code for an enzyme that can be used in food processing such as xylanases and xcex1-galactosidase. The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for xcex1-amylase, a protease or a glucanase. Alternatively, the GOI can be a nucleotide sequence according to the present invention.
The GOI can be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of our co-pending UK patent application 9505479.7. The GOI can be the nucleotide sequence coding for the glucanase enzyme which is the subject of our co-pending UK patent application 9505475.5. The GOI can be the nucleotide sequence coding for the xcex1-amylase enzyme which is the subject of our co-pending UK patent application 9413439.2. The GOI can be the nucleotide sequence coding for the xcex1-amylase enzyme which is the subject of our co-pending UK patent application 9421290.9. The GOI can be any of the nucleotide sequences coding for the a-glucan lyase enzyme which are described in our co-pending PCT patent application PCT/EP94/03397.
In one aspect the GOI can even be a nucleotide sequence according to the present invention but when operatively linked to a different promoter.
The GOI could include a sequence that codes for one or more of a xylanase, an arabinase, an acetyl esterase, a rhamnogalacturonase, a glucanase, a pectinase, a branching enzyme or another carbohydrate modifying enzyme or proteinase. Alternatively, the GOI may be a sequence that is antisense to any of those sequences.
As mentioned above, the present invention provides a mechanism for selectively affecting a particular enzymatic activity. In an important application of the present invention it is now possible to reduce or eliminate expression of a genomic nucleotide sequence coding for a genomic protein or enzyme by expressing an antisense intron construct for that particular genomic protein or enzyme and (e.g. at the same time) expressing a recombinant version of that enzyme or proteinxe2x80x94in other words the GOI is a recombinant nucleotide sequence coding for the genomic enzyme or protein. This application allows expression of desired recombinant enzymes and proteins in the absence of (or reduced levels of) respective genomic enzymes and proteins. Thus the desired recombinant enzymes and proteins can be easily separated and purified from the host organism. This particular aspect of the present invention is very advantageous over the prior art methods which, for example, rely on the use of anti-sense exon expression which methods also affect expression of the recombinant enzyme.
Thus, a further aspect of the present invention relates to a method of expressing a recombinant protein or enzyme in a host organism comprising expressing a nucleotide sequence coding for the recombinant protein or enzyme; and expressing a further nucleotide sequence wherein the further nucleotide sequence codes, partially or completely, for an intron in an antisense orientation; wherein the intron is an intron normally associated with the genomic gene encoding a protein or an enzyme corresponding to the recombinant protein or enzyme; and wherein the further nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron. Additional aspects cover the combination of those nucleotide sequences including their incorporation in constructs, vectors, cells, tissues and transgenic organisms.
Therefore the present invention also relates to a combination of nucleotide sequences comprising a first nucleotide sequence coding for a recombinant enzyme; and a second nucleotide sequence which corresponds to an intron in antisense orientation. wherein the intron is an intron that is associated with a genomic gene encoding an enzyme corresponding to the recombinant enzyme; and wherein the second nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron.
The GOI may even code for one or more introns, such as any one or more of the intron sequences presented in the attached sequence listings. For example, the present invention also covers the expression of for example an antisense intron (e.g. the complement of SEQ. ID. No. 38) in combination with for example a sense intron which preferably is not complementary to the antisense intron sequence (e.g. SEQ.I.D.No. 2 or another class A SBE intron).
The terms xe2x80x9ccellxe2x80x9d, xe2x80x9ctissuexe2x80x9d and xe2x80x9corganxe2x80x9d include cell, tissue and organ per se and when within an organism.
The term xe2x80x9corganismxe2x80x9d in relation to the present invention includes any organism that could comprise the nucleotide sequence according to the present invention and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism. Preferably the organism is a starch producing organism such as any one of a plant, algae, fungi, yeast and bacteria, as well as cell lines thereof. Preferably the organism is a plant.
The term xe2x80x9cstarch producing organismxe2x80x9d includes any organism that can biosynthesise starch. Preferably, the starch producing organism is a plant.
The term xe2x80x9cplantxe2x80x9d as used herein includes any suitable angiosperm, gymnosperm, monocotyledon and dicotyledon. Typical examples of suitable plants include vegetables such as potatoes; cereals such as wheat, maize, and barley; fruit; trees; flowers; and other plant crops. Preferably, the term means xe2x80x9cpotatoxe2x80x9d.
The term xe2x80x9ctransgenic organismxe2x80x9d in relation to the present invention includes any organism that comprises the nucleotide sequence according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the nucleotide sequence of the present invention is incorporated in the genome of the organism. Preferably the transgenic organism is a plant, more preferably a potato.
To prepare the host organism one can use prokaryotic or eukaryotic organisms. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press).
Even though the enzyme according to the present invention and the nucleotide sequence coding for same are not disclosed in EP-B-0470145 and CA-A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention. Some of these background teachings are now included in the following commentary.
The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
Several techniques exist for inserting the genetic information. the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
Thus, in one aspect, the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
The vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheuna An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D. N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208. Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.
The nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appears to be essential for insertion of modified T-DNA into the plant genome.
As will be understood from the above explanation, if the organism is a plant the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
Furthermore, the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof. As these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
In the construction of a transgenic plant the nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant. An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used. When a vector of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacteriun tumefaciens. The Ti-plasmid harboring the nucleotide sequence or construct of the present invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the present invention, which DNA is subsequently transferred into the plant cell to be modified.
If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley, et at., Crit. Rev. Plant Sci., 4:1-46: and An et al., EMBO J. (1985) 4:277-284.
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D. N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). With this technique, infection of a plant may be performed in or on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
Typically, with direct infection of plant tissues by Agrobacterium carrying the GOI (such as the nucleotide sequence according to the present invention) and, optionally, a promoter, a plant to be infected is wounded, e.g. by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
Further teachings on plant transformation may be found in EP-A-0449375.
As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 322, pUC series, M13 mp series, pACYC 184 etc. In this way, the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is then used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
After the introduction of the nucleotide sequence or construct according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessaryxe2x80x94such as to create combination systems as outlined above (e.g. an organism comprising a combination of constructs).
The above commentary for the transformation of prokaryotic organisms and plants with the nucleotide sequence of the present invention is equally applicable for the transformation of those organisms with the promoter of the present invention.
In summation, the present invention relates to affecting enzyme activity by expressing antisense intron sequences.
Also, the present invention relates to a promoter useful for the expression of those antisense intron sequences.
The following samples have been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on Jul. 13, 1995:
NCIMB 40753 (which refers to pBEA 8 as described herein);
NCIMB 40751 (which refers to xcex-SBE 3.2 as described herein), and
NCIMB 40752 (which refers to xcex-SBE 3.4 as described herein).
The following sample has been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on Jul. 9, 1996:
NCIMB 40815 (which refers to pBEA 9 as described herein).
A highly preferred embodiment of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed; and wherein the nucleotide sequence is antisense to intron 1 of class A SBE as set forth in SEQ. ID. No. 38, or any other intron of class A SBE, including fragments thereof, and including combinations of class A antisense intron sequences and class B antisense intron sequences. The sequence of introns of class A SBE other than intron 1 may be obtained by sequencing of, for example, potato class A SBE genomic DNA, isolatable by hybridisation screening of a genomic DNA library with class A SBE cDNA obtainable according to W096/34968 according to methods well known in the art and set forth, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989.